The journey of cardosin A in young Arabidopsis seedlings leads to evidence of a Golgi-independent pathway to the protein storage vacuole.

The aspartic proteinase cardosin A is a vacuolar enzyme found to accumulate in protein storage and lytic vacuoles in the flowers and protein bodies in the seeds of the native plant cardoon. Cardosin A was first isolated several decades ago and has since been extensively characterized, both in terms of tissue distribution and enzyme biochemistry. In the native system, several roles have been attributed to cardosin A, such as reproduction, reserve mobilization, and membrane remodeling. To participate in such diverse events, cardosin A must accumulate and travel to different compartments within the cell: protein storage vacuoles, lytic vacuoles, and the cytoplasmic membrane (and eventually outside the cell). Several studies have approached the expression of cardosin A in Arabidopsis thaliana and Nicotiana tabacum with promising results for the use of these systems to study of cardosin A trafficking. A poly-sorting mechanism has been uncovered for this protein, as two different vacuolar sorting determinants, mediating different vacuolar routes, have been described. The first is a conventional C-terminal domain, which delivers the protein to the vacuole via the Golgi, and the second is a more unconventional signal-the plant-specific insert (PSI)-that mediates a Golgi-independent route. The hypothesis that these two signals are activated according to cell needs and in organs with high metabolic activity is investigated here. An Arabidopsis line expressing cardosin A under an inducible promoter was used to understand the dynamics of cardosin A regarding vacuolar accumulation during seed germination events. Using antibodies against different regions of the protein and combining them with immunofluorescence and immunocytochemistry assays in different young seedling tissues, cardosin A was detected along the secretory pathway to the protein storage vacuole, often associated with the endoplasmic reticulum. More interestingly, upon treatment with the drug Brefeldin A, cardosin A was still detected in protein storage vacuoles, indicating that the intact protein can bypass the Golgi in this system, contrary to what was observed in N. tabacum. This study is a good starting point for further research involving the use of fluorescent fusions and exploring in more detail the relationship between cardosin A trafficking and plant development.

Coping with Abiotic Stress in Plants-An Endomembrane Trafficking Perspective.

Plant cells face many changes through their life cycle and develop several mechanisms to cope with adversity. Stress caused by environmental factors is turning out to be more and more relevant as the human population grows and plant cultures start to fail. As eukaryotes, plant cells must coordinate several processes occurring between compartments and combine different pathways for protein transport to several cellular locations. Conventionally, these pathways begin at the ER, or endoplasmic reticulum, move through the Golgi and deliver cargo to the vacuole or to the plasma membrane. However, when under stress, protein trafficking in plants is compromised, usually leading to changes in the endomembrane system that may include protein transport through unconventional routes and alteration of morphology, activity and content of key organelles, as the ER and the vacuole. Such events provide the tools for cells to adapt and overcome the challenges brought on by stress. With this review, we gathered fragmented information on the subject, highlighting how such changes are processed within the endomembrane system and how it responds to an ever-changing environment. Even though the available data on this subject are still sparse, novel information is starting to untangle the complexity and dynamics of protein transport routes and their role in maintaining cell homeostasis under harsh conditions.

Relevance of the Exocyst in Arabidopsis exo70e2 Mutant for Cellular Homeostasis under Stress.

Plants must adapt to cope with adverse environmental conditions that affect their growth and development. To overcome these constraints, they can alter their developmental patterns by modulating cellular processes and activating stress-responsive signals. Alongside the activation of the antioxidant (AOX) system, a high number of genes are expressed, and proteins must be distributed to the correct locations within the cell. The endomembrane system and associated vesicles thus play an important role. Several pathways have been associated with adverse environmental conditions, which is the case for the exocyst-positive organelle-EXPO. The present work, using Arabidopsis mutants with T-DNA insertions in the gene EXO70, essential for EXPO vesicles formation, was designed to characterise the anatomical (morphology and root length), biochemical (quantification of stress markers and antioxidant system components), and molecular responses (gene expression) to abiotic stresses (saline, drought, oxidative, and metal-induced toxicity). The results obtained showed that mutant plants behave differently from the wild type (WT) plants. Therefore, in the exo70 mutant, morphological changes were more noticeable in plants under stress, and the non-enzymatic component of the antioxidant system was activated, with no alterations to the enzymatic component. Furthermore, other defence strategies, such as autophagy, did not show important changes. These results confirmed the EXPO as an important structure for tolerance/adaptation to stress.

Abiotic Stress Triggers the Expression of Genes Involved in Protein Storage Vacuole and Exocyst-Mediated Routes.

Adverse conditions caused by abiotic stress modulate plant development and growth by altering morphological and cellular mechanisms. Plants' responses/adaptations to stress often involve changes in the distribution and sorting of specific proteins and molecules. Still, little attention has been given to the molecular mechanisms controlling these rearrangements. We tested the hypothesis that plants respond to stress by remodelling their endomembranes and adapting their trafficking pathways. We focused on the molecular machinery behind organelle biogenesis and protein trafficking under abiotic stress conditions, evaluating their effects at the subcellular level, by looking at ultrastructural changes and measuring the expression levels of genes involved in well-known intracellular routes. The results point to a differential response of the endomembrane system, showing that the genes involved in the pathway to the Protein Storage Vacuole and the exocyst-mediated routes are upregulated. In contrast, the ones involved in the route to the Lytic Vacuole are downregulated. These changes are accompanied by morphological alterations of endomembrane compartments. The data obtained demonstrate that plants' response to abiotic stress involves the differential expression of genes related to protein trafficking machinery, which can be connected to the activation/deactivation of specific intracellular sorting pathways and lead to alterations in the cell ultrastructure.

N-Linked Glycosylation Modulates Golgi-Independent Vacuolar Sorting Mediated by the Plant Specific Insert.

In plant cells, the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant-Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSI domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlike its native glycosylated form, is able to bypass ER-to-Golgi blockage and accumulate in the vacuole. This is also true when the PSI domain is analyzed in the context of the full-length cardosin. Regardless of opening exciting research gaps, the results obtained so far need a more comprehensive study of the mechanisms behind this unconventional direct sorting to the vacuole.

Vacuolar Sorting Determinants: Isolation and Study.

As it serves an important function in the compartmentalization of a series of components, from secondary metabolites to proteins, the vacuole is a central organelle in plant cell biology and development. One of the most important mechanisms regulating not only vacuolar biogenesis but also its luminal content, is the vacuolar sorting of proteins. This sorting mechanism is based upon the recognition of specific signal sequences, vacuolar sorting determinants, by sorting receptors, which then act by redirecting vacuolar cargo away from the default secretory pathway, and into the vacuolar lumen. One of the most direct ways of understanding if a given peptide possesses vacuolar sorting determinant capability is to isolate it, fuse it with a marker and express it in a plant system such as Nicotiana tabacum, a pipeline which will be described in more detail in this chapter.

Zinc Accumulation and Tolerance in Solanum nigrum are Plant Growth Dependent.

Zinc tolerance, accumulation, and organic acid production by Solanum nigrum, a known Zn accumulator, was studied during pre- and post-flowering stages of development. The plants, when challenged with Zn concentrations lethal to plantlets, showed an increase in tolerance from pre-flowering to post-flowering, which was accompanied by a reduction of Zn translocation to the aerial plant parts. Treatment with Zn induced a differential response in organic acids according to the plant organ and developmental stage. In the roots, where Zn concentrations were similar in pre- and post-flowering plants, a general decrease in organic acid in pre-flowering roots contrasted with the increase observed in post-flowering plants. In the stems, Zn induced a generalized increase in organic acids at both growth stages while in the leaves, a slight increase in malic and shikimic was observed in pre-flowering plants and only shikimic acid levels were significantly increased in post-flowering plants. This work shows that Zn accumulation and tolerance in S. nigrum vary during plant development--an observation that may be important to improve the efficiency of phytoremediation approaches. Furthermore, the data suggest the involvement of specific organic acids in this response.

Histological and ultrastructural evidence for zinc sequestration in Solanum nigrum L.

The accumulation of contaminants in the environment due to anthropogenic activities is a matter of global concern. Solanum nigrum L. plants, able to accumulate zinc and hyperaccumulate cadmium, were challenged with 0.025 g Zn L(-1) during 35 days. The localization of Zn in roots, stems and leaves of S. nigrum plants was evaluated by autometallography (AMG) in order to determine the specific tissue, cell and subcellular compartments of Zn sequestration. This Zn concentration resulted in stunted plant growth but no other symptoms of Zn toxicity. Zinc concentration in the plants was highest in the roots, 666 mg Zn kg(-1) fresh weight (f.w.), and lower in the stems, 318 mg Zn kg(-1) f.w., and leaves, 101 mg Zn kg(-1) f.w. Roots of Zn-treated plants showed an underdeveloped structure but additional layers of proliferating cortical parenchyma cells. AMG of S. nigrum roots, stems and leaves revealed a generalized Zn distribution associated with the cell walls in all tissues. In the vasculature (xylem and phloem), Zn was observed at the plasma membrane-cell wall complex of vascular parenchyma cells and conducting elements. Conspicuous Zn deposits were detected in the vacuoles of cortical parenchyma and starch sheath, as well as in the tonoplast of the mesophyll cells. Our results suggest that Zn flux through the plant occurs via the xylem and phloem and associated parenchyma until it is conducted to permanent storage sites, namely, the apoplast and vacuoles of cortical parenchyma cells of the root, stem and the leaf mesophyll.

A putative role for γ-aminobutyric acid (GABA) in vascular development in pine seedlings.

MAIN CONCLUSION: A model for GABA synthesis in stems of pine seedlings is proposed. The localization of GABA in differentiating tracheids suggests a link between GABA production and vascular development. γ-aminobutyric acid (GABA) is a non-proteinogenic amino acid present in both prokaryotic and eukaryotic organisms. GABA plays a fundamental role as a signal molecule in the central nervous system in animals. In plants, GABA has been correlated with cellular elongation, plant development, gene expression regulation, synthesis of ethylene and other hormones, and signaling. Considering the physiological importance of GABA in plants, the lack of works about GABA localization in this kingdom seems surprising. In this work, the immunolocalization of GABA in root and hypocotyl during seedling development and in bent stem showing compression xylem has been studied. In the seedling root, the GABA signal was very high and restricted to the stele supporting previous evidences indicating a potential role for this amino acid in root growth and nutrient transport. In hypocotyl, GABA was localized in vascular tissues, including differentiating xylem, ray parenchyma and epithelial resin duct cells, drawing also a role for GABA in vascular development, communication and defense. During the production of compression wood, a special lignified wood produced when the stem loss its vertical position, a clear GABA signal was found in the new differentiating xylem cells showing a gradient-like pattern with higher signal in less differentiated elements. The results are in accordance with a previous work indicating that glutamate decarboxylase and GABA production are associated to vascular differentiation in pine Molina-Rueda et al. (Planta 232: 1471-1483, 2010). A model for GABA synthesis in vascular differentiation, communication, and defense is proposed in the stem of pine seedlings.

Metal uptake by native plants and revegetation potential of mining sulfide-rich waste-dumps.

Waste dumps resulting from metal exploitation create serious environmental damage, providing soil and water degradation over long distances. Phytostabilization can be used to remediate these mining sites. The present study aims to evaluate the behavior of selected plant species (Erica arborea, Ulex europaeus, Agrostis delicatula, and Cytisus multiflorus) that grow spontaneously in three sulfide-rich waste-dumps (Lapa Grande, Cerdeirinha, and Penedono, Portugal). These sites represent different geological, climatic and floristic settings. The results indicate distinctive levels and types of metal contamination: Penedono presents highest sulfate and metal contents, especially As, with low levels of Fe. In contrast, at Lapa Grande and Cerdeirinha Fe, Mn, and Zn are the dominant metals. In accordance, each waste dump develops a typical plant community, providing a specific vegetation inventory. At Penedono, Agrostis delicatula accumulates As, Pb, Cu, Mn, and Zn, showing higher bioaccumulation factors (BF) for Mn (32.1) and As (24.4). At Cerdeirinha, Ulex europaeus has the highest BF for Pb (984), while at Lapa Grande, Erica arborea presents high BF for Mn (9.8) and Pb (8.1). Regarding TF, low values were obtained for most of the metals, especially As (TF < 1). Therefore, the results obtained from representative plant species suggest appropriate behavior for phytostabilization measures.

Delivering of proteins to the plant vacuole--an update.

Trafficking of soluble cargo to the vacuole is far from being a closed issue as it can occur by different routes and involve different intermediates. The textbook view of proteins being sorted at the post-Golgi level to the lytic vacuole via the pre-vacuole or to the protein storage vacuole mediated by dense vesicles is now challenged as novel routes are being disclosed and vacuoles with intermediate characteristics described. The identification of Vacuolar Sorting Determinants is a key signature to understand protein trafficking to the vacuole. Despite the long established vacuolar signals, some others have been described in the last few years, with different properties that can be specific for some cells or some types of vacuoles. There are also reports of proteins having two different vacuolar signals and their significance is questionable: a way to increase the efficiency of the sorting or different sorting depending on the protein roles in a specific context? Along came the idea of differential vacuolar sorting, suggesting a possible specialization of the trafficking pathways according to the type of cell and specific needs. In this review, we show the recent advances in the field and focus on different aspects of protein trafficking to the vacuoles.

Cardosin A contains two vacuolar sorting signals using different vacuolar routes in tobacco epidermal cells.

Several vacuolar sorting determinants (VSDs) have been described for protein trafficking to the vacuoles in plant cells. Because of the variety in plant models, cell types and experimental approaches used to decipher vacuolar targeting processes, it is not clear whether the three well-known groups of VSDs identified so far exhaust all the targeting mechanisms, nor if they reflect certain protein types or families. The vacuolar targeting mechanisms of the aspartic proteinases family, for instance, are not yet fully understood. In previous studies, cardosin A has proven to be a good reporter for studying the vacuolar sorting of aspartic proteinases. We therefore propose to explore the roles of two different cardosin A domains, common to several aspartic proteinases [i.e. the plant-specific insert (PSI) and the C-terminal peptide VGFAEAA] in vacuolar sorting. Several truncated versions of the protein conjugated with fluorescent protein were made, with and without these putative sorting determinants. These domains were also tested independently, for their ability to sort other proteins, rather than cardosin A, to the vacuole. Fluorescent chimaeras were tracked in vivo, by confocal laser scanning microscopy, in Nicotiana tabacum cells. Results demonstrate that either the PSI or the C terminal was necessary and sufficient to direct fluorescent proteins to the vacuole, confirming that they are indeed vacuolar sorting determinants. Further analysis using blockage experiments of the secretory pathway revealed that these two VSDs mediate two different trafficking pathways.

Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii.

Aspartic proteinases have been extensively characterized in land plants but up to now no evidences for their presence in green algae group have yet been reported in literature. Here we report on the identification of the first (and only) typical aspartic proteinase from Chlamydomonas reinhardtii. This enzyme, named chlapsin, was shown to maintain the primary structure organization of typical plant aspartic proteinases but comprising distinct features, such as similar catalytic motifs DTG/DTG resembling those from animal and microbial counterparts, and an unprecedentedly longer plant specific insert domain with an extra segment of 80 amino acids, rich in alanine residues. Our results also demonstrated that chlapsin accumulates in Chlamydomonas chloroplast bringing this new enzyme to a level of uniqueness among typical plant aspartic proteinases. Chlapsin was successfully expressed in Escherichia coli and it displayed the characteristic enzymatic properties of typical aspartic proteinases, like optimum activity at acidic pH and complete inhibition by pepstatin A. Another difference to plant aspartic proteinases emerged as chlapsin was produced in an active form without its putative prosegment domain. Moreover, recombinant chlapsin showed a restricted enzymatic specificity and a proteolytic activity influenced by the presence of redox agents and nucleotides, further differentiating it from typical plant aspartic proteinases and anticipating a more specialized/regulated function for this Chlamydomonas enzyme. Taken together, our results revealed a pattern of complexity for typical plant aspartic proteinases in what concerns sequence features, localization and biochemical properties, raising new questions on the evolution and function of this vast group of plant enzymes.

The heterologous systems in the study of cardosin B trafficking pathways.

Cardosins are abundant in cardoon pistils and were found to accumulate in different cell compartments: cardosin A was detected in the vacuoles of stigmatic papillae and cardosin B accumulates in the extracellular matrix of the transmitting tissue. Due to the fact that cardoon system imposes some limitations to the study of processing and trafficking events, heterologous species have been employed to study cardosins trafficking pathways. Cardosin B was successfully expressed both in Arabidopsis and Tobacco plants, where it accumulated mainly in the vacuole but it was also detected in the cell wall. The glycosylation pattern of cardosin B was replicated in these systems - high-mannose type glycans. In tobacco leaves, cardosin B is transported through the Golgi in a RAB-D2a-dependent route, and is delivered to the vacuole via the prevacuolar compartment in a RAB-F2b-dependent pathway. 

Insights into phytoremediation solutions for environmental recovery.

Our environment is contaminated with organic and inorganic compounds released by anthropogenic activities that cause negative impacts on biological productivity and ecosystem sustainability and place human health at risk. Within the available remediation technologies, phytoremediation has emerged with high potential due to its reduced environmental impacts and economic costs. The research into phytoremediation has developed through a wide array of approaches, which also pertains to its inherent interdisciplinary characteristics, towards enhancing the potential of the technology for application in the field. Numerous patents present molecular solutions through which plants can be engineered to display improvements in key characteristics, such as the tolerance, uptake and accumulation of contaminants. The manipulation of plant growth and of the physico-chemical characteristics of the contaminated environments in order to enhance the remediation potential has also been the focus of several issued patents. This review attempts to highlight the most relevant patented advances in phytoremediation and to emphasise recent research efforts through which this green technology might be expected to develop into a commercially competitive alternative to other remediation methods.

Dissecting cardosin B trafficking pathways in heterologous systems.

In cardoon pistils, while cardosin A is detected in the vacuoles of stigmatic papillae, cardosin B accumulates in the extracellular matrix of the transmitting tissue. Given cardosins' high homology and yet different cellular localisation, cardosins represent a potentially useful model to understand and study the structural and functional plasticity of plant secretory pathways. The vacuolar targeting of cardosin A was replicated in heterologous species so the targeting of cardosin B was examined in these systems. Inducible expression in transgenic Arabidopsis and transient expression in tobacco epidermal cells were used in parallel to study cardosin B intracellular trafficking and localisation. Cardosin B was successfully expressed in both systems where it accumulated mainly in the vacuole but it was also detected in the cell wall. The glycosylation pattern of cardosin B in these systems was in accordance with that observed in cardoon high-mannose-type glycans, suggesting that either the glycans are inaccessible to the Golgi processing enzymes due to cardosin B conformation or the protein leaves the Golgi in an early step before Golgi-modifying enzymes are able to modify the glycans. Concerning cardosin B trafficking pathway, it is transported through the Golgi in a RAB-D2a-dependent route, and is delivered to the vacuole via the prevacuolar compartment in a RAB-F2b-dependent pathway. Since cardosin B is secreted in cardoon pistils, its localisation in the vacuoles in cardoon ovary and in heterologous systems, suggests that the differential targeting of cardosins A and B in cardoon pistils results principally from differences in the cells in which these two proteins are expressed.

Processing and trafficking of a single isoform of the aspartic proteinase cardosin A on the vacuolar pathway.

Cardosin A is the major vacuolar aspartic proteinase (APs) (E.C.3.4.23) in pistils of Cynara cardunculus L. (cardoon). Plant APs carry a unique domain, the plant-specific-insert (PSI), and a pro-segment which are separated from the catalytic domains during maturation but the sequence and location of processing steps for cardosins have not been established. Here transient expression in tobacco and inducible expression in Arabidopsis indicate that processing of cardosin A is conserved in heterologous species. Pulse chase analysis in tobacco protoplasts indicated that cleavage at the carboxy-terminus of the PSI could generate a short-lived 50 kDa intermediate which was converted to a more stable 35 kDa intermediate by removal of the PSI. Processing intermediates detected immunologically in tobacco leaves and Arabidopsis seedlings confirmed that cleavage at the amino-terminus of the PSI either preceded or followed quickly after cleavage at its carboxy-terminus. Thus removal of PSI preceded the loss of the prosegment in contrast to the well-characterised barley AP, phytepsin. PreprocardosinA acquired a complex glycan and its processing was inhibited by brefeldin A and dominant-inhibitory AtSAR1 or AtRAB-D2(a )mutants indicating that it was transported via the Golgi and that processing followed ER export. The 35 kDa intermediate was present in the cell wall and protoplast culture medium as well as the vacuole but the 31 kDa mature subunit, lacking the amino-terminal prosegment, was detected only in the vacuole. Thus maturation appears to occur only after sorting from the trans-Golgi to the vacuole. Processing or transport of cardosin A was apparently slower in tobacco protoplasts than in whole cells.

EDDS and EDTA-enhanced zinc accumulation by Solanum nigrum inoculated with arbuscular mycorrhizal fungi grown in contaminated soil.

The effect of two different chelating agents [EDTA and EDDS (S,S-ethylenediaminedissucinic acid)] on Zn tissue accumulation in Solanum nigrum L. grown in a naturally contaminated soil was assessed. Under those conditions, the response of the plant to the inoculation with two different isolates of arbuscular mycorrhizal fungi (AMF)--Glomus claroideum and Glomus intraradices--was also studied. Plants grown in the local contaminated soil (Zn levels of 433mg kg(-1)) accumulated up to 1191mg kg(-1) of Zn in the roots, 3747mg kg(-1) in the stems and 3409mg kg(-1) in the leaves. S. nigrum plants grown in the same soil spiked with extra Zn (Zn levels of 964mg kg(-1)) accumulated up to 4735, 8267 and 7948mg Zn kg(-1) in the leaves, stems and roots, respectively. The addition of EDTA promoted an increase in the concentration of Zn accumulated by S. nigrum of up to 231% in the leaves, 93% in the stems and 81% in the roots, while EDDS application enhanced the accumulation in leaves, stems and roots up to 140, 124 and 104%, respectively. In the stems, the presence of Zn was predominantly detected in the cortex collenchyma cells, the starch sheath and the internal phloem and xylem parenchyma, and the addition of chelating agents did not seem to have an effect on the localisation of accumulation sites. The devise of a chelate-enhanced phytoextraction strategy, using chelating agents and AMF, is discussed.

Solanum nigrum grown in contaminated soil: effect of arbuscular mycorrhizal fungi on zinc accumulation and histolocalisation.

Zn tissue accumulation in Solanum nigrum grown in a non-contaminated and a naturally contaminated Zn matrix and the effect of inoculation with different arbuscular mycorrhizal fungi (AMF) on metal uptake were assessed. S. nigrum grown in the contaminated soil always presented higher Zn accumulation in the tissues, accumulating up to 1622 mg Zn kg(-1). The presence of both Glomus claroideum and Glomus intraradices enhanced the uptake and accumulation of Zn by S. nigrum (up to 83 and 49% higher Zn accumulation, respectively). The main deposits of the metal were found in the intercellular spaces and in the cell walls of the root tissues, as revealed by autometallography, with the inoculation with different AMF species causing no differences in the location of Zn accumulation. These findings indicate that S. nigrum inoculated with selected heavy metal tolerant AMF presents extracting and accumulating capacities, constituting a potentially suitable remediation method for Zn polluted soils.